Optimal conditions for protein array deposition using continuous flow.
Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.
Natarajan
Sriram
S
Department of Chemical Engineering, University of Utah, Salt Lake City, Utah 84112, USA.
Hatch
Andrew
A
Myszka
David G
DG
Gale
Bruce K
BK
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000915:Antibody Affinity; D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D002417:Cattle; D055808:Drug Discovery; D040081:Protein Array Analysis / Q000379:methods*; D013205:Staphylococcal Protein A / Q000378:metabolism; D013997:Time Factors
D000906:Antibodies; D013205:Staphylococcal Protein A; D001360:Avidin